The role of integrins in cancer and the development of anti-integrin therapeutic agents for cancer therapy.

Integrins have been reported to mediate cell survival, proliferation, differentiation, and migration programs. For this reason, the past few years have seen an increased interest in the implications of integrin receptors in cancer biology and tumor cell aggression. This review considers the potential role of integrins in cancer and also addresses why integrins are present attractive targets for drug design. It discusses of the several properties of the integrin-based chemotherapeutic agents currently under consideration clinically and provides an insight into cancer drug development using integrin as a target.


Introduction
Integrins are a large family of eukayotic cell-surface receptors that mediate dynamic interaction between cells and extracellular adhesion molecules (Humphries, 2000). The integrins recognize extracellular matrix (ECM) proteins or counter-receptors on adjacent cells. ECM molecules that affect cell adhesion include glycoproteins such as fi bronectin (Fn) (Gardner and Hynes, 1985), von Willebrand factor (vWF) (Chow et al. 1992), vitronectin (Vn) (Pytela et al. 1985), thrombospondin (Tsp) (Karczewski et al. 1989), tenascin (Tn) (Joshi et al. 1993), collagen (Coll) (Heino, 2000), laminin (Ln) (Burgeson and Christiano, 1997), osteopontin (Opn) (Green, 2001), and other unidentifi ed molecules. A key fi nding in the discovery of the integrins was that the well known amino acid sequence Arg-Gly-Asp (RGD) which was initially found in fi bronectin, serves as a primary cell recognition motif. Subsequently, the RGD sequence was found in many ECM molecules and, in many cases, was responsible for cell attachment (Karczewski et al. 1989;Joshi et al. 1993;Ruggeri et al. 1983;Davis, 1992;Schnapp et al. 1995;Kimura et al. 1998). The recent crystal structures of the extracellular domains of α V β 3 (Xiong et al. 2001; have provided new insights into integrin activation and ligand recognition. The interaction of integrins with their ligands is dependent upon signals transduced from the cytoplasmic tails to the extracellular domains (Travis et al. 2003). The binding of integrins to their ligands is critically important to many diverse physiological phenomena, such as attachment, cell proliferation (Miyata et al. 2000;Hollenbeck et al. 2004;Hedin et al. 2004;Zhou et al. 2004), migration (Hirsch et al. 1996;Sakai et al. 1998;Fujiwara et al. 2001;Paulhe et al. 2001). Integrins also contribute to the initiation and/or progression of many diseases including tumor invasion, angiogenesis and metastasis (Tsuji et al. 2004;Takanami et al. 2005;Guo et al. 2005;Enserink et al. 2004;Chung et al. 2004;Felding-Habermann et al. 2002;Gladson et al. 1996;Zheng et al. 1999;Zheng et al. 2000).

Integrin Structure
The first three-dimensional structure of the extracellular domain of an integrin was published in October 2001, a decade and a half after the family was fi rst defi ned (Xiong et al. 2001(Xiong et al. , 2002(Xiong et al. , 2004 (Fig. 1). Crystal structure of integrin α V β 3 showing the dimer and individual subunits (Xiong et al. 2002). An unliganded ectodomain from the αA-lacking integrin α V β 3 contains the two subunts assembled into a globular head built by two predicted domains: the N-terminal seven-bladed propeller domain of α V and an αA-like domain (βA) from the β 3 . βA loops out from the "Hybrid" domain (β 3 residues 55-108 and 353-432), which itself is inserted in the N-terminal plexin/semaphorin/integrin (PSI) domain (residues 1-54 and residues 433-435) of β 3 . The PSI domain and the beta-tail domain (βTD), together forming the β 3 leg. Ig-like thigh domain and calf-1 and calf-2 domains formed the α V leg. Two legs are bent at the "knees" and folded back against the head of the same molecule. This sharp bending takes place between the thigh and calf-1 of α V (α-genu) and approximately corresponding to between EGF domains 1 and 2 of β 3 (β-genu). A metal ion (Ca 2+ or Mn 2+ ) occupies the α-genu on both the ligand and unliganded structures. At the base of propeller, blades 4-7 each contain a metal ion coordinated in a β-hairpin loop.

Integrin and Cancer
Cancer occurs when cells become abnormal and keep dividing and forming more cells without control or order. If cells keep dividing when new cells are not needed, a mass undifferentiated tissue forms. This mass of extra tissue, called a growth or tumour, can constitute either a benign or a malignant tumour respectively. Benign tumors can usually be removed and, in most cases, they do not come back. Most importantly, cells from benign tumors do not spread to other parts of the body. Benign tumors are rarely a threat to life. In contrast, malignant tumours are truly cancerous. Cancer cells can invade and damage nearby tissues and organs. Cancer cells can break away from a malignant tumor and enter the bloodstream or the lymphatic system. The spread of cancer is called metastasis which appears to be a complex multistep process that involves the invasion of cancer cells from primary neoplasm followed by their dissemination through the lymphatic vessels and systemic circulation. New blood vessels form either by vasculogenesis, which refers to initial events of vascular growth in which endothelial cell precursors (angioblasts) differentiate and assemble into primitive vessels or by angiogenesis, which refers to a combination of sprouting of new vessels from pre-existing ones, and longitudinal separation of pre-existing vessels in a process named intussusception (Conway et al. 1993). The angiogenesis can be triggered in pathological conditions such as tumor growth and chronic wounding. Angiogenetic process involves functional cooperativity between cytokines and endothelial cell (EC) surface integrins. Cell bound integrins by their physical interaction with ligands necessary are essential for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. They also trigger a variety of signal transduction pathways which are involved in mediating invasion, metastasis and squamous-cell carcinoma which can be reviewed as follows. The review focuses mainly on specifi c α and β subtypes which have been most extensively investigated in cancer.

β 1 class of integrins
Although little clear correlation between tumor formation, invasion and β 1 integrin expression has yet been demonstrated in human patients, it has been possible to show a crucial role of β 1 integrin in tumor formation and metastasis in mice. Tumor cells expressing β 1 integrin formed signifi cantly larger primary tumors and had a dramatically increased metastasis into liver and lung (Brakebusch et al. 1999). In another study, which used a T cell lymphoma line in which both β 1 integrin alleles was deleted by homologous recombination, metastasis formation in mice was signifi cantly reduced (Stroeken et al. 2000). Recently, it was shown that ablation of the β 1 integrin gene in mammary epithelium dramatically impaired mammary tumorigenesis in mice (White et al. 2004). Sudhakar et al. have reported that human collagen α1(IV)NC1 binds to α 1 β 1 integrin, competes with type IV collagen binding to α 1 β 1 integrin, and inhibits migration, proliferation, and tube formation by ECs, indicating that α1 (IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis (Sudhakar et al. 2005). A study using β 1 integrin double knockout lymphocytes and retransfection of β 1 integrin deletion mutants have shown that different parts of the cytoplasmic domain of β 1 integrin are required either for adhesion or for invasion and metastasis (Stroeken et al. 2000).
Integrin α 1 β 1 and α 2 β 1 were shown to regulate hepatocarcinoma cell invasion across the fi brotic matrix microenvironment (Yang et al. 2003). A potent selective inhibitor of α 1 β 1 integrin, obtustatin purifi ed from the venom of the Vipera lebetina obtusa viper was reported to have a marked ability to inhibit angiogenesis in vivo in the chicken chorioallantoic membrane assay, and in the Lewis lung syngeneic mouse model (Marcinkiewicz et al. 2003). Grzesiak and Bouvet have demonstrated that the certain cancer cell lines including CFPAC (a ductal epithelioid cell line established from a cystic fi brosis patient with pancreatic adenocarcinoma), BxPC-3 (human pancreas adenocarcinoma), Colo-357 (human lymph node metastasis), and Panc-1 (Pancreatic Cancer Cell Line) attach to 3D type I collagen scaffolds in an α 2 β 1 -specifi c manner and that this integrin-specifi c adhesion is required for subsequent cell proliferation. Such evidences support the notion that targeting α 2 β 1 integrin-specifi c type I collagen adhesion may have therapeutic value in the treatment of pancreatic cancer (Grzesiak and Bouvet, 2007). Integrin α 2 β 1 was also reported to mediate the anti-angiogenic and anti-tumor activities of angiocidin, a novel tumour-associated protein which is capable of binding to both α 2 β 1 and type I collagen. This protein promoted α 2 β 1 -dependent cell adhesion and inhibited tumor growth and angiogenesis . Combined antagonism of α 1 β 1 and α 2 β 1 was shown to reduce tumor growth substantially as well as angiogenesis of human squamous cell carcinoma xenografts (Senger et al. 2002).
The interaction of α 3 β 1 with ligand laminin-5 has been demonstrated to promote the migration and invasion of malignant glioma and melanoma cells (Tsuji, 2004;Tsuji et al. 2002;Giannelli et al. 2007) and to promote binding to virus glycoprotein. A signifi cant increase in proliferation and adhesion in response to collagen 1 and laminin for integrin receptor α 3 β 1 was also observed in ovarian cancer cell lines (Ahmed et al. 2005). More recently, uPAR (urokinase-type plasminogen activator receptor), and TIMP (tissue inhibitors of metalloproteinases)-2 were also proposed as ligands of α 3 β 1 integrin in mediating uPA/uPAR interaction and intracellular signaling (Wei et al. 2007). In an animal model it was shown that soluble uPAR antagonizes cancer progression (Jo et al. 2003).
The Src family kinases are classifi ed as oncogenic proteins due to their ability to activate cell migration (Rodier et al. 1995;Rahimi et al. 1998) in many cell types including epithelial tumor cells. Studies with chimeric α 4 integrin subunits have shown that α 4 cytoplasmic domain can enhances cell migration via c-Src activation (Chan et al. 1992;Hsia et al. 2005).
α 5 β 1 integrin interacts with Fn which is implicated in several cellular activities including cell proliferation, differentiation, and migration. A high-affi nity interaction that occurs with the central cell binding domain, a region involved in many fundamental aspects of cell growth and morphogenesis, is dependent on the RGD sequence and other recognition sequences (Li et al. 2003;Murillo et al. 2004). The interaction with Fn has been demonstrated with both lung epithelial cells and fi broblasts. In addition, the inhibition of cell surface α 5 integrin expression was found to decrease phosphoinositide-3 kinase (PI3K) activity and inhibit colon cancer cell attachment, suggesting that agents which selectively target α 5 integrin subunit expression may enhance the effects of standard chemotherapeutic agents and provide a novel adjuvant treatment for selected colon cancers (Lopez-Conejo et al. 2002). Furthermore, cells expressing the α 5 β 1 integrin displayed a dramatic enhancement in the ability of growth factors to activate PI3K and protein kinase B (PKB), indicating this stimulation may also involve the interaction between α 5 β 1 and the PI3 K and PKB signalling pathways . Wei et al. recently reported that urokinase receptor binding to α 5 β 1 is required for maximal responses to Fn and tumor cell invasion (Wei et al. 2007). Kuwada et al. demonstrated that expression of integrin α5β1 in colon cancer cells decreases HER (human epidermal growth factor receptor)-2-mediated proliferation, crystal violet assays were showing inhibition of the cell proliferation of Caco-2 control cells with the antagonistic HER-2 antibody mAb 4D5 (Kuwada et al. 2005). MAb 4D5 is also indicated clinically active in cancer patients to target HER2-overexpression (Baselga et al. 1996;Rhodes, 2005). Furthermore, mAb 4D5 has been shown great promise as targeted agents in the treatment of patients with cancer (Bartsch et al. 2007).
It has been reported that α 6 integrin-mediated neutrophil migration through the perivascular basement membrane (PBM) is platelet-endothelial cell adhesion molecule1 (PECAM-1) dependent, a response associated with PECAM-1-mediated increased expression of α 6 β 1 on transmigrating neutrophils (Dangerfi eld et al. 2002). Signifi cantly increased ovarian cancer cell line proliferation and adhesion to collagen 1 and laminin (ligands of integrin receptor α 6 β 1 ) were also reported (Ahmed et al. 2005). In addition, an α 6 integrin is found to be overexpressed in human oesophageal carcinomas, suggesting an important role in oesophageal tumor invasion (Tanaka et al. 2000). This notion has since been confi rmed by other studies (Mercurio et al. 2001;Demetriou et al. 2004).
The α 7 β 1 integrin is a laminin-binding receptor that was originally identified in melanoma (Kramer et al. 1991). Ziober et al. reported that during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the α 7 β 1 (Ziober et al. 1999). Integrin α 7 β 1 serves an important mechanical function in the diaphragm by contributing to passive compliance, viscoelasticity, and modulation of muscle contractile properties (Lopez et al. 2005).
Integrin α 10 β 1 is a major collagen-binding integrin during cartilage development and in mature hyaline cartilage while α 11 β 1 was originally found in fetal muscle (Gullberg et al. 1995). Integrin α 11 β 1 recognizes the triple-helical GFOGER sequence (where single letter amino acid nomenclature is used, O = hydroxyproline) found in interstitial collagens (Tulla et al. 2001). Little is known about the biology of these recently identifi ed integrins. Integrin α 10 β 1 is expressed on chondrocytes and some fi brous tissues. Integrin α 11 β 1 is involved in cell migration and collagen reorganization in mesenchymal non-muscle cells (Tiger et al. 2001).
Recently, α 11 β 1 integrin is required on periodontal ligament fi broblasts for cell migration and collagen reorganization by assisting axial tooth movement (Popova et al. 2007).

α V class of integrin
The fi rst integrin found associated with tumor angiogenesis was α V β 3 (Eliceiri et al. 1998;Eliceiri, 2001;Ruegg et al. 2003). Integrin α V β 3 has a broad distribution and is found on endothelial cells, smooth muscle cells (SMCs) and hematopoietic cell types such as platelets and osteoclasts. The interaction of α V β 3 with its ligands plays a crucial role in angiogenesis and neointimal formation after vascular injury. In addition, during osteoclast-mediated bone resorption, α V β 3 regulates the cytoskeletal organization required for cell migration and formation of the sealing zone (McHugh et al. 2000). Prostate cancer specifi c integrin α V β 3 was demonstrated to modulate bone metastatic growth and tissue remodeling (McCabe et al. 2007). The study of co-expression of bone sialoprotein, integrin α V β 3 , and MMP-2 in papillary thyroid carcinoma cells demonstrated that cancer cells appear to become more invasive when bone sialoprotein forms a cell-surface trimolecular complex that links MMP-2 to integrin α V β 3 (Karadag et al. 2004). Bayless et al. present very convincing data showing that integrin α V β 3 as well as integrin α 5 β 1 regulate human endothelial cell vacuolation and lumen formation, implicating a major role contributed by these two integrins for endothelial cell morphogenesis (Bayless et al. 2000). It is also clear that the integrin α V β 3 plays an important role in virtually every stage of cancer progression. Indeed, neuroblastoma aggressiveness has been identifi ed to be correlated with the expression of integrin α V β 3 and α V β 5 by microvascular endothelium (Erdreich-Epstein et al. 2000). Other studies also demonstrate that increased α V β 3 expression level is closely associated with increased cell invasion and metastasis (Feldin-Habermann et al. 2002). Li et al. reported that antisense α V suppressed tumour growth more strongly than antisense β 3 , antisense therapy but simultaneous targeting at both integrin subunits was more effective than the respective monotherapies (Li et al. 2007). Integrin α V β 3 has been demonstrated to interact with the activated forms of the platelet-derived growth factor, insulin, and vascular endothelial growth factor (VEGF) cell receptors faciliting optimal activation of cell proliferative signalling pathways (Giancotti and Ruoslahti, 1999;Kumar, 2003). The functional activity of αvβ3 on endothelial and tumor cells may well be regulated by VEGF (Byzova et al. 2000). VEGF has been also implicated in prostate carcinogenesis and metastasis as well as in angiogenesis. Both VEGF and its receptor are expressed by prostate carcinoma cells at a high level (Ferrer et al. 1998;. A role for α V β 6 -mediated production in the regulation of MMP-9 and MMP-3 have been reported in several tumor types and in untransformed keratinocytes (Ramos et al. 2002;Ahmed et al. 2002). MMP-9 plays a critical role in the recruitment of bone marrow derived CD45 positive cells into the primary tumor and the establishment of a mature vasculature (Jodele et al. 2005). Integrin α V β 6 also plays a role in wound healing and cancer of the oral cavity (Thomas et al. 2006). In addition, α V β 6 has been implicated in the regulatory control of the uPA proteolytic cascade (Ahmed et al. 2002). A gradual increase in the expression of α V β 6 integrin from borderline to malignant tumors has been reported in oral squamous carcinomas (Jones et al. 1997) and breast carcinomas (Arihiro et al. 2000). In malignant keratinocytes and colon cancer cells, increased expression of this integrin enhances MMP-9 secretion and MMP-9-mediated invasion (Thomas et al. 2000;Agrez et al. 1999). Inhibition of α V β 6 function using inhibitory antibodies results in total abrogation of MMP-9 activation (Thomas et al. 2001) suggesting that the expressions of α V β 6 integrin and MMP-9 are linked, and their coordinate expression appears to promote invasion by squamous and colon carcinoma cells. The integrin α V β 6 interacts with Fn, Vn , tenascin (Weinacker et al. 1995), and latencyassociated peptide (Munger et al. 1999), a protein derived from the N-terminal region of the transforming growth factor(TGF)-β gene product that mediates cell adhesion, spreading, migration, proliferation, and activation of latent TGF-β (Weinreb et al. 2004;Thomas et al. 2001).
Until recently, there has been little information about integrin α V β 8 which has been reported to function as an additional receptor for foot-andmouth disease virus (FMDV) (Jackson et al. 2004) in addition to the three RGD-dependent integrins α V β 1 , α V β 3 , and α V β 6 , which have been shown to be receptors for FMDV previously (Jackson et al. 1997(Jackson et al. , 2000(Jackson et al. , 2002Duque et al. 2004). Notably, α V β 8 as well as α V β 6 may promote epithelialmesenchymal transition (EMT) by contributing to the activation of TGF-β (Munger et al. 1999). Additionally, α V β 8 -mediated activation of TGF-β was shown to block the proliferation of certain cancer cells (Mu et al. 2002). Several recent studies have demonstrated that both up-regulation and down-regulation of expression of α V integrins and other integrins can be effective markers of malignant diseases and patient prognosis.
Although there are few reports of enhanced expression of α IIb β 3 (than of α V β 3) integrin in tumour cells, one observation indicated an important role in tumour progression. A study on human melanoma biopsies showed that α IIb β 3 expression increased with tumour thickness (Trikha et al. 2002a). In addition, a single pretreatment of human melanoma cells with c7E3 Fab, an α IIb β 3 antibody inhibited lung colonization of the tumor cells in severe combined immunodefi cient mice (Trikha et al. 2002b).

Other sub-classes of integrins
Parathyroid hormone-related protein (PTHrP) was reported to not only increase transcriptional activity of the integrin subunit α 5 (Anderson et al. 2007) but also upregulate integrin α 6 β 4 expression and activate Akt in breast cancer cells (Dittmer et al. 2006;Shen and Falzon, 2006;Shen et al. 2007). Falcioni et al. fi rst identifi ed a tumor antigen (TSP-180) associated with metastasis that was shown to be identical to the β 4 integrin subunit (Falcioni et al. 1997;Kennel et al. 1989). Subsequently other studies showed that expression of α 6 β 4 persists in some aggressive carcinomas and that its expression may be linked to the behavior of these tumors (Guo et al. 2004). At earlier of the year in 2001, Davis and his colleagues demonstrated that α 6 β 4 integrin has an infl uence on tumour biology as this integrin and its ligand, laminin-5, are essential gene products for the maintenance and remodeling of a stratifi ed epithelium (Davis et al. 2001). The β 4 integrin, for example, was lost in the lesions of prostatic intraepithelial neoplasia together with basal cell-lining and in prostate carcinoma the expression of β 4 integrins was totally lost (Davis et al. 2001). In normal skin keratinocytes, expression of the α 6 β 4 integrin is restricted to the proliferative basal layer and mediates stable adhesion to the underlying basement membrane. Observations in carcinoma cells show a functional and spatial dissociation of the α 6 β 4 integrin from the hemidesmosomal complex, which stimulates cell migration and, therefore, may contribute to carcinoma invasion (Kippenberger et al. 2004). Indeed, many carcinomas express elevated levels of α 6 β 4 (Herold-Mende et al. 2001), particularly breast carcinomas (Chung and Mercurio, 2004). Pawar et al. recently have shown that the uPAmediated cell surface cleavage of the alpha6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin (Pawar et al. 2007). In addition, observations in carcinoma cells show a functional and spatial dissociation of the α 6 β 4 integrin from the hemidesmosomal complex, which stimulates cell migration (Kippenberger et al. 2004). Furthermore, blocking antibodies to either α 6 or β 4 integrin subunits suppress the formation of apoptosis-resistant acinar structures in Matrigel by mammary epithelial cells (Weaver et al. 2002), suggesting a role for β 4mediated cellular polarity in mediating antiapoptotic signaling. Integrin α 6 β 4 was recently noted only at the cell's basal interface with the basement membrane in normal pancreatic ducts. But in pancreatic adenocarcinomas, 92% demonstrated overexpression of integrin α 6 β 4 and altered localization to the cytoplasm and membranous regions, this upregulation and redistribution of integrin α 6 β 4 expression implicated a role of integrin in pancreatic adenocarcinoma progression (Cruz-Monserrate et al. 2007). Interestingly, the expression of β 4 was inversely correlated with dissemination of ten human gastric cancer cell lines in SCID (severe combined immunodeficiency) mice (Ishii et al. 2000). In addition, strong evidence suggests that reduced expression of α 6 and β 4 subunits may contribute to the higher tumorigenicity of androgen-independent prostate tumor cells (Bonaccorsi et al. 2000).
Several key signalling molecules in carcinoma cells are also involved in the mechanisms of α 6 β 4 integrin-mediated tumour behaviour Folgiero et al. 2007) since β 4 has been demonstrated to interact with ERBB2 (erythroblastic leukemia viral oncogene homolog 2, encoding an 185-kDa, 1255 amino acids, orphan receptor tyrosine kinase) that displays potent oncogenic activity when overexpressed) in some cultured breast tumour cells, and the two proteins synergize in promoting cellular proliferation and invasion (Falcioni et al. 1997). In addition, Guo et al. established that integrin α 6 β 4 may be required for mammary tumourigenesis driven by the expression of ErBB2 (Guo et al. 2006). Folgiero et al. revealed that α 6 β 4 can regulate the expression of ErBB-3 at the level of protein translation, resulting in a signifi cant induction of ErBB-2/ErBB-3 heterodimerization and consequent activation of PI3K Liu et al. 2007). Introduction of β 4 in β 4 -negative breast carcinoma cells activates signalling from PI3K to Rac (a member of the Rho family of small guanosine triphosphatases) and increases the invasion of these cells in vitro (Shaw, 2001).
The integrin α E β 7 (also known as cell marker CD103) is expressed by most intra-epithelial lymphocytes (IEL). An important ligand for this molecule is the epithelial cell adhesion molecule E-cadherin. Cresswell et al. have demonstrated that the up-regulation of integrin α E β 7 by lymphocytes increases adhesion to E-cadherin expressing bladder cancer targets, indicating a role of integrin α E β 7 in cancer invasion (Cresswell et al. 2002).

Integrins as Targets for the Treatment of Cancer
From what has been discussed above, integrins play a key role in tumor angiogenesis and cancer. Because they are cell surface receptors interacting with extracellular ligands, they represent ideal pharmacological targets. A variety of integrin antagonists such as low molecular weight inhibitors, peptidomimetics, or monoclonal antibodies are in various stages of development as anti-cancer therapeutics (Kerr et al. 2002;Mousa 2002;Tucker et al. 2003).
In-vivo study has demonstrated that the addition of inhibitory anti-β 1 -integrin antibodies or the re-expression of α 2 β 1 integrins leads to the reversal of the malignant phenotype in a 3-dimensional cell culture model and to a reduction in tumour formation in animal models (Zutter et al. 1995). Yao et al. recently show that β 1 integrin expression has potential prognostic value in invasive breast cancer and that coexpression of fi bronectin may help identify patients with more aggressive tumors who may benefi t from targeted therapy (Yao et al. 2007). More studies have been focused on α V β 3 , since α V β 3 has been identifi ed as a prognostic indicator of survival and a specifi c potential target for control of angiogenesis, therapies directed against integrin against α V β 3 , have been developed (Brooks et al. 1994;Gladson et al. 1996;Zhang et al. 2007;Gramoun et al. 2007).

MEDI-552
Brooks and his coworkers first showed that a monoclonal antibody specifi c for α V β 3 , MEDI-552 (LM609), could block angiogenesis in a murine model (Brooks et al. 1994). In addition, there is an ongoing phase I dose escalation study evaluating the safety of MEDI-522 in patients with advanced malignancies. This antibody was chosen for its unique ability to selectively target multiple and different cell types. In a phase I trial on various solid tumours, MEDI-522 appeared to be without signifi cant toxicity (McNeel et al. 2005). MEDI-522 was detectable both in quiescent and in angiogenically active skin blood vessels as well as in the dermal interstitial space. The levels of phosphorylated focal adhesion kinase (pFAK) were reduced during MEDI-522 treatment, suggesting a modulating effect on this signaling molecule Gramoun et al. 2007).

CNTO 95
A fully humanized monoclonal antibody to anti-α V integrins, CNTO 95, has been shown to inhibit angiogenesis and tumor growth in preclinical studies (Mullamitha et al. 2007). CNTO 95 is likely to be less immunogenic in humans compared to chimeric or humanized antibodies (Trikha et al. 2004). CNTO 95 bound to purifi ed α V β 3 and α V β 5 with higher affi nity (a Kd of approximately 200 pM and to α V integrin-expressing human cells with a Kd of 1-24 nM). In vitro, CNTO 95 potentially inhibited human melanoma cell adhesion, migration and invasion (doses ranging 7-20 nM) and appeared to be safe without inhibition of normal physiologic angiogenesis (Martin et al. 2005;Trikha et al. 2004).

17E6
The 17E6 antibody strongly perturbs cell attachment mediated by α V associated integrins, by reacting with α V β 3 , α V β 5 , and α V β 1 , and has the ability to disrupt stable interaction between vitronectin and α V β 3 , and blocks the growth of M21 tumours in nude mice. In two nude mouse tumor models, injection of 17E6 strongly inhibited tumor development (Mitjans et al. 2000;Mitjans et al. 1995).
Integrin antibodies that block specifi c integrins for treatment of cancer are still in clinical trial stages as lessons should be learnt from integrin antibodies for the treatment of other diseases. For example, Tysabri (also called natalizumab), an antibody which blocks α 4 integrins and inhbits the α 4 -mediated adhesion of leukocytes to their counterreceptor (s) (Minagar et al. 2000;O'Connor et al. 2004O'Connor et al. , 2005. Although the specific mechanism(s) by which tysabri exerts its effects in multiple sclerosis (MS) have not been fully characterized, Tysabri was initially approved by the Food and Drug Administration (FDA) in U.S.A. in November, 2004 for the treatment of patients with relapsing forms of MS, but was withdrawn by the manufacturer three months later after three patients developed progressive multifocal leukoencephalopathy (PML), a serious viral infection of the brain, in the drug's clinical trials, FDA then put clinical trials of the drug on hold, allowing them to resume a year later after confi rming that there were no additional cases of PML. In June 2006, the FDA resumed marketing of Tysabri with a restricted distribution program. Tysabri is indicated for use as monotherapy, because we do not know enough about how its use with other immune modifying drugs could impact risk. (www.fda. gov/cder/drug/infopage/natalizumab).
Other antibodies α 1 β 1 and α 2 β 1 integrins play a signifi cant role in the VEGF-driven angiogenesis. Ha 31/8 and Ha 1/29 are antibodies against α 1 and α 2 integrin subunits which were reported to inhibit endothelial cells in a gradient of immobilized collagen I assay (haptotaxis) by Ͻ40%, whereas the combination of both antibodies synergized to reach Ͻ90% inhibition (Alghisi and Ruegg, 2006). Consistent with these results, administration of both the antiα 1 and the anti-α 2 antibodies to nude mice bearing a human A431 squamous cell carcinoma xenograft suppressed angiogenesis by Ͻ60% and tumor growth by Ͼ40% (Senger et al. 2002). Interestingly, preclinical studies with monoclonal antibodies (MAbs) against lactadherin, a glycoprotein of the milk fat globule membrane was found that there was a clear increase in VEGF-like proangiogenic activity (Taylor et al. 1997;Silvestre et al. 2005) when lactadherin is added back exogenously to the ischemic muscles. An investigation has further identifi ed lactadherin as a physiological ligand of α V β 3 and α V β 5 , thus confi rming a proangiogenic activity of these integrins in the VEGF-dependent neovascularization in adult mice, but not in embryos. Animal test showed that the expression of Flk-1 (VEGFR-2) is elevated in β 3 -defi cient mice, indicating that α V β 3 can control the amplitude of the VEGF response by controlling the Flk-1 level or activity (Reynolds et al. 2004). In vitro, anti-α 5 β 1 function-blocking mAbs (NKI-SAM-1, JBS5, or IIA1) inhibited adhesion in a 72% to 100% range depending on the cell line used. This result was further confi rmed in vivo in an angiogenesis assay treated with fi broblast growth factor 2 (Kim et al. 2000). The anti-α 5 β 1 M200 antibody (Volociximab) is another chimeric monoclonal antibody of α 5 β 1 integrin that blocks tumor growth and metastasis. M200 binds to α 5 β 1 integrin on activated endothelial cells with high binding affi nity and inhibits in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of α 5 β 1 function by M200 induced apoptosis of actively proliferating, but not resting endothelial cells (Ramakrishnan et al. 2006).

Disintegrins, RGD-Based Peptides and Small Molecule Integrin Antagonists
The "disintegrin" terminology was initially applied in 1990 to describe a family of cysteine-rich, RGDcontaining proteins from viper venom toxins that inhibit platelet aggregation and integrin-mediated cell adhesion (Gould et al. 1990;Niewiarowski et al. 1994;McLane et al. 2004). Studies of RGDcontaining proteins in venom toxins have been found that a number of them, such as contortrostatin, salmosin and bitistatin (Markland et al. 2001;Zhou et al. 1999;Swenson et al. 2004;Golubkov et al. 2003;Kang et al. 1999;Chung et al. 2003;McQuade et al. 2004), are able to inhibit tumor growth and angiogenesis. Echistatin has been found to induce a decrease of both autophosphorylation and kinase activity of pp125FAK, suggesting inhibitory activity in processes integral to angiogenesis, such as cell growth, survival, and migration (Della et al. 2000). Trifl avin was found to interact with either α IIb β 3 on platelet membranes, resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, or α V β 3 on SMCs subsequently inhibiting cell migration and proliferation (Sheu et al. 2001). Trifl avin also blocks neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury (Fu et al. 2004). Recently, soluble RGD peptides have been demonstrated to induce apoptosis by inducing conformational changes in procaspases, leading to increased oligomerization and subsequent autoprocessing of these enzymes (Buckley et al. 1999). In addition, RGD-containing proteins from venom toxins (e.g. salmosin, contortrostatin, rhodostomin and accutin) were also found to induce apoptosis (Chung et al. 2003;Zhou et al. 1999;Wu et al. 2003;Yeh et al. 1998). It is still not clear whether these proteins' apoptotic induction is through interaction with integrins or through a different apoptotic pathway since Jan et al. in a recent issue of Cell have shown that integrins may regulate apoptosis, through caspase-independent mechanisms (Jan et al. 2004). These data have shown the potential for these RGD-containing snake venom proteins to function as integrin antagonists as well as antiangiogenic and antimetastatic compounds, leading to drug development for therapeutic usage (Markland et al. 2001;Kerr et al. 2002;Hallahan et al. 2001;Coller, 2001).
The integrins that bind to RGD peptides are generally over-expressed in angiogenic vessels. In certain cancer, the tumor cells also express RGDbinding integrins. A vast body of preclinical and clinical literature exists on the use of RGD-based integrin antagonists in cardiovascular disease and cancer (Tucker, 2003;McQuade and Knight, 2003;Kumar et al. 2003;Shimaoka and Springer, 2004). A cyclic pentapeptide called EMD66203 [cyclic L-Arg-L-Gly-L-Asp-D-Phe-L-Val (RGDfV) peptide or cyclo(-Arg-GlyAsp-D-Phe-Val)] (Aumailley et al. 1991) was shown preferential inhibition of vitronectin binding to the α V β 3 rather than to the α IIb β 3 (Frieser et al. 1996). Further modifi cation of the EMD66203 led to the synthesis of EMD121974, an RGD-containing pseudopeptide (c(RGDfV)) or cyclo(Arg-Gly-Asp-D-Phe-[NMe]Val) also known as cilengitide (Dechantsreiter et al. 1999). Structural study revealed that the D-amino acid in this peptide is found preferentially in position i + 1 of a β II' turn, a characteristic for its biological activity. EMD121974 is also a dual α V β 3 /α V β 5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy (Belvisi et al. 2005(Belvisi et al. , 2006. The crystal structure of the extracellular segment of integrin α V β 3 in complex with EMD121974 revealed that the pentagonal peptide inserted into a crevice between the propeller and βA domains on the integrin head (Xiong et al. 2002). EMD 121974 was demonstrated to be an α V -integrin antagonist and a potent inhibitor of angiogenesis, by inducing apoptosis of growing endothelial cells through inhibition of their α V -integrin interaction with the matrix proteins vitronectin and tenascin (Taga et al. 2002).
ST1646, an RGD-containing pseudopeptide, is a potent, highly selective α V β 3 /α V β 5 integrin antagonist, equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) (Belvisi et al. 2006;Haier et al. 2002). The structure docking model for the ST1646-α V β 3 complex has confi rmed that, similarly to the crystal structure of the EMD121974-α V β 3 complex, the ligand seems to interact mainly through electrostatic forces in a rather shallow cleft and that essentially no hydrophobic interactions can be observed (Belvisi et al. 2005). In an in vitro anti-angiogenic activity assay, ST1646 inhibited HUVEC proliferation with potency similar to EMD121974 (IC 50 , 2.9 and 4.4 µmol/L for the two compounds, respectively). The inhibitory effect was reversible. In an in vivo antiangiogenic activity assay as determined by daily administration of ST1646 (30 µg/embryo) with CAM (chick chorioallantoic membrane) assay at day 9 via a gelatin sponge implant and at day 12 for histologic analysis, showed signifi cant inhibition of the angiogenic response triggered by both FGF2 and VEGF (p Ͻ 0.001) (Belvisi et al. 2005).
SCH 221153, an RGD-based peptidomimetic, inhibits the binding of the disintegrin, echistatin to α V β 3 and α V β 5 with similar potency, according to IC 50 values of 3.2 nM and 1.7 nM, respectively (Kumar et al. 2001). SCH 221153 inhibits FGF2 and VEGF-induced endothelial cell proliferation in vitro according to IC 50 equal to 3-10 µM (Kumar et al. 2001). Monsanto-Searle (St. Louis, MO) has reported an orally compound SC-68448 which inhibited α V β 3 -mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. SC68448 was 100-fold more potent as a functional inhibitor of α V β 3 versus α IIb β 3 (Carron et al. 2000). Integrin α IIB β 3 expressed mainly on platelet membrane plays a crucial role in platelet aggregation and thrombus formation, and recently was reported to have a role in increasing the risk of metastases in renal cell carcinoma in men (Kallio et al. 2006). Haubner and his co-workers reported that 18 F-Galacto-RGD is a highly α V β 3 -selective tracer for positron emission tomography (PET) (Haubner et al. 2004(Haubner et al. , 2001. Molecular imaging with 18 F-Galacto-RGD and PET provides important information for planning and monitoring anti-angiogenic therapies targeting the α V β 3 integrin (Beer et al. 2006). Meerovitch et al. demonstrated BCH-14661 and BCH-15046, RGD peptidomimetic compounds are as apoptotic inducers for endothelial cells by causing cell detachment-dependent when cells are grown on RGD-containing integrin ligand vitronectin and fi bronectin. BCH-14661 was specifi c for integrin α V β 3 , whereas BCH-15046 nonselectively antagonized α V β 3 , α V β 5 , and α 5 β 1 (Meerovitch et al. 2003). A 20 amino acid N-terminal peptide of angiocidin was reported to promote α 2 β 1 -dependent adhesion of K562 cells, disrupt human umbilical vein endothelial cell tube formation and inhibit tumour growth as well as angiogenesis in a mouse model . Angiocidin has also been reported to inhibit angiogenesis through binding collagen and integrin α 2 β 1 present on many tumour cells .
The most selective nonpeptidic α 5 β 1 antagonist SJ749 showed a reduced proliferation of astrocytoma cell lines dependent on α 5 β 1 expression levels and cell culture conditions, underlining the importance of α 5 β 1 as a target for anticancer therapies (Marinelli et al. 2005;Maglott et al. 2006). A non-peptide RGD mimetic, S36578-2, was also developed and demonstrated as highly selective antagonist of both α V β 3 and α V β 5 integrins that was able to induce detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin (Maubant et al. 2006). Reinmuth and his co-works demonstrated that S-247, another α V β 3 /α V β 5 integrin antagonist, showed signifi cant antimetastatic and antiangiogenic activity and impaired both endothelial and hVSMC/pericyte function in vitro and in vivo (Reinmuth et al. 2003;Harms et al. 2004).
The integrin-induced signaling cascades have also been demonstrated to impact tumor cell survival, cell migration, and angiogenesis. It is known that transforming growth factor (TGF)-beta suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-beta, thus enabling TGF-beta to promote the acquisition and development of metastatic phenotypes. TGF-β stimulation can induce α V β 3 integrin expression in a manner that coincides with epithelialmesenchymal transition (EMT) in MECs. Introduction of siRNA against β 3 integrin can block TGF-β induction and also prevent TGF-β stimulation of EMT in MECs (Galliher and Schiemann, 2006). Therefore, antagonists of growth factor receptors (Cardones et al. 2006;Wick et al. 2006) can be used for anti-cancer therapy. Indeed, the recognition of potent, sequence-selective gene inhibition by siRNA oligonucleotides and rapid adoption as the tool of choice in cell culture has generated the expectation for their use to improve targeted therapeutics (Elbashir et al. 2002;Paddison et al. 2003;Carpenter and Sabatini, 2004;Ganju and Hall, 2004). The prospects of siRNA to be a therapeutic tool were enhanced by their double-stranded RNA (dsRNA) oligonucleotide nature, resembling antisense, ribozymes and gene therapy (Song et al. 2003;Davidson et al. 2004). Silencing integrin α V expression by siRNA can inhibit proliferation and induce apoptosis in integrin α V over-expressing MDA-MB-435 human breast cancer cells (Cao et al. 2006). Lipscomb and his co-workers demonstrated that siRNA oligonucleotides targeted to either subunit of the α 6 β 4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells (Lipscomb et al. 2003). Recently gene transfer of antisense α V and β 3 expression vectors was demonstrated to downregulate α V and β 3 in HepG2 tumours established in nude mice, inhibit tumour vascularization and growth, and enhance tumour cell apoptosis, suggesting that antisense gene therapy targeting α V integrins could be as an approach to treat hepatocellular carcinomas (Li et al. 2007).
A study using SUM-159 breast carcinoma cell line showed that decreased expression of the α 6 β 4 integrin led to enhanced apoptosis. Recombinant VEGF is able to signifi cantly inhibit the cell death observed in the β 4 -defi cient cell line. The specifi city of α 6 β 4 in both in vitro and in vivo assays showed that reexpression of the β 4 subunit into the β 4 -defi cient cell line could rescue the functional phenotype (Lipscomb et al. 2005).

Conclusions
In this review the potential roles of integrin in tumor progression and cancer were discussed. Evidence presented here indicates that integrins represent highly appropriate pharmacological targets as based upon the benefi cial effect of integrin antibodies and antagonists in cancer treatment. A number of the integrin antibodies and antagonists are now in clinical trials, determining their effect on angiogenesis, metastasis and tumour growth (Table 2).  Reinmuth et al. 2003;Harms et al. 2004